ABSTRACT
The deep cerebellar nuclei (DCN) integrate various inputs to the cerebellum and form the final cerebellar outputs critical for associative sensorimotor learning. However, the functional relevance of distinct neuronal subpopulations within the DCN remains poorly understood. Here, we examined a subpopulation of mouse DCN neurons whose axons specifically project to the ventromedial (Vm) thalamus (DCNVm neurons), and found that these neurons represent a specific subset of DCN units whose activity varies with trace eyeblink conditioning (tEBC), a classical associative sensorimotor learning task. Upon conditioning, the activity of DCNVm neurons signaled the performance of conditioned eyeblink responses (CRs). Optogenetic activation and inhibition of the DCNVm neurons in well-trained mice amplified and diminished the CRs, respectively. Chemogenetic manipulation of the DCNVm neurons had no effects on non-associative motor coordination. Furthermore, optogenetic activation of the DCNVm neurons caused rapid elevated firing activity in the cingulate cortex, a brain area critical for bridging the time gap between sensory stimuli and motor execution during tEBC. Together, our data highlights DCNVm neurons' function and delineates their kinematic parameters that modulate the strength of associative sensorimotor responses.
Subject(s)
Animals , Mice , Blinking , Cerebellar Nuclei/physiology , Cerebellum , Neurons/physiology , ThalamusABSTRACT
ObjectiveTo investigate the molecular mechanism of cordycepin inhibiting proliferation and promoting apoptosis of human hepatoma cells （HCCs）. MethodGlioma-associated oncogene homolog 1 （Gli1） gene was silenced by small interfering RNA （siRNA） and transfected into SMMC-7721 cells， and then cell proliferation was detected by cell counting kit-8 （CCK-8） assay and cell cloning assay. SMMC-7721 cells were treated with different concentration of cordycepin， and the cell proliferation and apoptosis were examined. The expression of Gli1 and the downstream related genes was determined by Real-time polymerase chain reaction（PCR） and Western blot. ResultThe mRNA and protein expression of Gli1 in SMMC-7721 cells was higher than that in normal liver cells （P<0.01）. The proliferation rate of SMMC-7721 with silenced Gli1 decreased at 72 and 96 h （P<0.05， P<0.01）， and the colony-forming capacity lowered （P<0.01） compared with those in the blank group. Compared with the control， 80 μmol·L-1 and 120 μmol·L-1 cordycepin significantly inhibited the proliferation of SMMC-7721 cells at 72 and 96 h （P<0.05， P<0.01）， and promoted the apoptosis of them （P<0.01）. Moreover， 80 and 120 μmol·L-1 cordycepin restrained the mRNA and protein expression of Gli1 in SMMC7721 cells （P<0.05， P<0.01）. At 120 μmol·L-1， cordycepin led to the decrease in the mRNA and protein levels of B-cell lymphoma-2 （Bcl-2） and c-Myc （P<0.05， P<0.01）， and the increase in the mRNA and protein expression of cysteine-aspartic acid protease-3 （Caspase-3） （P<0.05）. ConclusionGli1 is highly expressed in HCCs， and cordycepin can suppress the proliferation and enhance the apoptosis of HCCs by regulating Gli1 and the downstream apoptosis-related factors.
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OBJECTIVE@#To explore the effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism.@*METHODS@#Thirty-six male ICR mice were randomly divided into three groups (=12):sham group, TCP wear particles (TCP) group and N-acetyl-L-cysteine (NAC) group. Aperiprosthetic osteolysis model in mouse was established by implanting 30 mg of TCP wear particles onto the surface of bilateral parietal bones following removal of the periosteum. On the 2nd day post-operation, NAC (1.0 mg/kg) was locally injected to the calvarium under the periosteum every other day for 2 weeks. Then, all the mice were sacrificed to obtain blood and the calvaria. Periprosthetic osteolysis in the mouse calvaria was observed by tartrate resistant acid phosphatase (TRAP) staining; serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6); total anti-oxidation capacity (T-AOC) and superoxide dismutase (SOD) activity were examined by ELISA and chemical colorimetry, respectively; protein levels of glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α) and phospho-eIF2α (p-eIF2α) in periprosthetic bone tissue were detected by Western blot.@*RESULTS@#Compared with sham group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were increased obviously in TCP group (<0.05), and serum level of T-AOC and SOD activity were decreased significantly in TCP group (<0.05), GRP78 expression, the ratio of p-PERK and PERK, p-eIF2α and eIF2α in the mouse calvaria of TCP group were up-regulated markedly. Compared with TCP group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were decreased markedly in NAC group (<0.05), serum level of T-AOC and SOD activity were increased obviously in NAC group (<0.05), and GRP78 expression, the ratio of p-PERK/PERK and p-eIF2α/eIF2α were obviously down-regulated.@*CONCLUSIONS@#Inhibition of oxidative stress can prevent periprosthetic osteolysis induced by TCP wear particles, which may be mediated by inactivation of PERK/eIF2α signaling pathway.
Subject(s)
Animals , Male , Mice , Mice, Inbred ICR , Osteolysis , Oxidative Stress , Skull , Tumor Necrosis Factor-alphaABSTRACT
AIM: To evaluate intraocular pressure (IOP) control and visual rehabilitation after placement of the Ex-press(R)miniature glaucoma shunt with adjunctive amniotic membrane transplantation (AMT) and mitomycin C (MMC) in patients with post-traumatic open-angle glaucoma during 2y of follow up.METHODS: This was an interventional,2-year,observational study.Eighteen eyes were prospectively observed (in 18 patients with traumatic secondary open-angle glaucoma) in which Ex-press miniature glaucoma filtration shunts were implanted with AMT and MMC.The outcome measures included intraocular pressure (IOP),best corrected visual acuity (BCVA),number of antiglaucoma medications,and complications.The progress of all patients was monitored for 24mo.RESULTS: Complete success (IOP <21 mmHg without glaucoma medications) was seen in 15 of the 17 (88.2%) eyes enrolled in the study at 24mo after the operation.IOP decreased from 36.9±4.8 mmHg preoperatively to 15.4±3.5 mmHg at 12mo and 15.5±3.5 mmHg at 24mo postoperatively.Early postoperative hypertension developed in two patients (11.1%) due to postoperative fibrosis.Most of the patients had improved postoperative BCVA values at the final follow-up visit compared to their preoperative measurements.Two patients (11.1%) developed transient hypotony.There were no complications such as hyphema,choroidal effusion,shallow anterior chamber,the device touching the iris,or extrusion of the device.CONCLUSION: The Ex-press miniature glaucoma filtration shunt with adjunctive AMT and MMC is effective and safe in cases of traumatic open-angle glaucoma.Surgical management is an appropriate surgical treatment in this series of cases.
ABSTRACT
Background:Dysregulation of microRNA expression is involved in the carcinogenesis of many tumors,expression of miR-504 has been observed in many tumors,but its expression in primary hepatocellular carcinoma(HCC)has not been reported. Aims:To explore the expression and clinical significance of miR-504 in HCC tissues and cells. Methods:Tumor and para-cancerous tissue in 85 HCC patients were collected. Five liver cancer cell lines and immortalized liver cell line were conventionally cultured. Effect of miR-504 expression on HepG2 cells proliferation was determined by CCK8 assay. The expression of miR-504 was determined by real time fluorescent quantitative PCR. Relationships between expression of miR-504 and clinicopathological features and prognosis of HCC patients were analyzed. Results:Compared with normal liver cells,expression of miR-504 was significantly downregulated in five liver cancer cells(P < 0. 05). Up-regulation of miR-504 inhibited proliferation of HepG2 cells,and down-regulation of miR-504 promoted proliferation of HepG2 cells. miR-504 expression in tumor tissue was significantly decreased than that in corresponding para-cancerous tissue. Expression of miR-504 was correlated with tumor differentiation(P = 0. 002),TNM stage(P = 0. 021),alpha fetoprotein(P = 0. 012)and portal vein tumor thrombus(P = 0. 003),but not with gender,age,tumor size and tumor number. Survival in HCC patients with lower miR-504 expression was significantly shorter than that in patients with high expression(P < 0. 05). Conclusions:Expression of miR-504 is downregulated in HCC,and is correlated with the prognosis of patients,suggesting that miR-504 can be used as an independent predictor for prognosis of HCC patients.
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miR-146a is an immunoregulatory microRNA closely associated with viral infection. This study investigated the expression changes of miR-146a in peripheral blood monocytes of HCV-infected patients and the mechanism by which the THP-1 cells were stimulated with HCV core protein in vitro. It was found that in the peripheral blood monocytes of HCV-infected patients, miR-146a expression was upregulated. After treated by interferon/ribavirin, miR-146a expression was decreased when HCV RNA became undetectable. HCV core could directly stimulate THP-1 cells to produce miR-146a. Silencing TLR2 and MyD88 could significantly inhibit the expression of miR-146a. It was concluded that the expression of miR-146a in peripheral blood monocytes of HCV-infected patients was abnormally increased. The TLR2-MyD88 signaling pathway may take part in the overexpression of miR-146a in monocytes stimulated with HCV core protein.
ABSTRACT
miR-146a is an immunoregulatory microRNA closely associated with viral infection. This study investigated the expression changes of miR-146a in peripheral blood monocytes of HCV-infected patients and the mechanism by which the THP-1 cells were stimulated with HCV core protein in vitro. It was found that in the peripheral blood monocytes of HCV-infected patients, miR-146a expression was upregulated. After treated by interferon/ribavirin, miR-146a expression was decreased when HCV RNA became undetectable. HCV core could directly stimulate THP-1 cells to produce miR-146a. Silencing TLR2 and MyD88 could significantly inhibit the expression of miR-146a. It was concluded that the expression of miR-146a in peripheral blood monocytes of HCV-infected patients was abnormally increased. The TLR2-MyD88 signaling pathway may take part in the overexpression of miR-146a in monocytes stimulated with HCV core protein.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Young Adult , Base Sequence , Cell Line , DNA Primers , Hepatitis C, Chronic , Blood , MicroRNAs , Blood , Monocytes , Metabolism , Myeloid Differentiation Factor 88 , Physiology , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 2 , PhysiologyABSTRACT
The activation of hepatic stellate cells (HSCs) and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-β (TGFβ)/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations (0-10 μmol/L). Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFβ1, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different concentrations (1, 2, 3 μmol/L) or RPMI1640 alone (serving as control). The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC50 of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 μmol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased (P0.05). There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 (P<0.05). However, the expression of Smad7 protein was substantially increased when compared with the control group (P<0.05). It was concluded that the inhibition of TGFβ/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors (TGFβ1, Smad3) and promote the expression of anti-fibrosis factor (Smad7), suggesting that MG132 may become a potential therapeutic alternative for liver fibrosis.
ABSTRACT
The activation of hepatic stellate cells (HSCs) and their transformation to myofibroblasts are the key steps in the pathological progress of liver fibrosis. The transforming growth factor-β (TGFβ)/Smad pathway is involved in the proliferation and collagen synthesis of HSCs. This study aimed to examine the effect of the protease inhibitor MG132 on the signaling pathway of TGFβ/Smad in HSC-T6 cells and seek a novel therapeutic approach for liver fibrosis. The HSC-T6 cells were treated with MG132 at different concentrations (0-10 μmol/L). Cell proliferation was detected by MTT method. The mRNA and protein expression levels of TGFβ1, Smad3 and Smad7 were determined in HSC-T6 cells by real-time PCR and Western blotting, respectively, after treatment with MG132 at different concentrations (1, 2, 3 μmol/L) or RPMI1640 alone (serving as control). The results showed that MG132 could inhibit the proliferation of HSC-T6 cells in a dose-dependent manner, and the IC(50) of MG132 was 6.84 μmol/L. After treatment with MG132 at 1, 2 or 3 μmol/L for 24 h, the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased (P<0.05), but the Smad7 mRNA expression had no significant change (P>0.05). There was also a significant decrease in the protein expression level of TGF-β1 and Smad3 (P<0.05). However, the expression of Smad7 protein was substantially increased when compared with the control group (P<0.05). It was concluded that the inhibition of TGFβ/Smad pathway in HSC-T6 cells by MG132 can reduce the production of profibrosis factors (TGFβ1, Smad3) and promote the expression of anti-fibrosis factor (Smad7), suggesting that MG132 may become a potential therapeutic alternative for liver fibrosis.
Subject(s)
Animals , Rats , Cell Line , Leupeptins , Pharmacology , Protease Inhibitors , Pharmacology , Signal Transduction , Smad Proteins , Metabolism , Transforming Growth Factor beta , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the incidence of heparin-induced thrombocytopenia (HIT) in patients received unfractionated heparin (UFH) treatment, and explore the feasibility of monitoring HIT by platelet counts, as well as the significance of HIT-antibody test in HIT diagnosis.</p><p><b>METHODS</b>145 patients received UFH treatment in Vascular Surgery Department were studied. Before and after the UFH treatment, platelet counts, HIT-antibody ELISA test and heparin-induced platelet aggregation (HIPA) were tested.</p><p><b>RESULTS</b>Among the 145 patients, thrombocytopenia occurred in 40 (27.6%) cases, HIT-antibody ELISA test positive in 59 (40.7%) cases, HIPA test positive in 26 (17.9%) cases. The HIT was diagnosed in 24 (16.5%) cases, and heparin-induced thrombocytopenia and thrombosis (HITTS) occurred in 5 (3.4% in all cases, and 20.8% in HIT patients). In HIT patients, 15 patients (62.5%) were thrombocytopenia, HIT-antibody positive and HIPA test positive. Platelet counts in all of the 24 patients recovered to normal or level before UFH treatment in 3-6 days after heparin withdrawal therapy.</p><p><b>CONCLUSION</b>HIT can be early diagnosed by monitoring platelet counts, HIT-antibody ELISA test and HIPA test. Withdrawal of heparin therapy in time and use of alternative anticoagulant, HITTS rate might be expected to decline further.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Anticoagulants , Enzyme-Linked Immunosorbent Assay , Heparin , Platelet Aggregation , Platelet Count , Thrombocytopenia , DiagnosisABSTRACT
The objective of this study was to investigate the immunophenotypic subtype profiles of 207 pediatric patients with acute lymphoblastic leukemia (ALL) and its correlation with cytogenetics and clinical features. 207 children with ALL were immunophenotyped by four color flow cytometry using a panel of monoclonal antibodies. 207 patients were enrolled in this study, out of which 146 cases were subjected to karyotype analysis by R-banding technology. The results showed that 11.6% out of 207 children with ALL were identified as T-ALL, 88.4% as B-ALL. Myeloid antigen (MyAg) expression was documented in 42.5% out of 207 cases analyzed and CD13 was the most commonly expressed MyAg (31.4%). No difference was observed in the expression of MyAg between the groups of patients with T-ALL (41.7%) and B-ALL (42.6%). Abnormal karyotypes were detected in 84 out of 146 (57.5%) children. The clinical and biological characteristics of ALL patients between MyAg(+) and MyAg(-) groups showed that higher percentage of patients with high WBC count (> 50 × 10(9)/L) and higher CD34 positivity were found to be correlated with MyAg(+) ALL. It is concluded that immunophenotype analysis is useful for ALL diagnosis and classification, and the immunophenotypes are in relevance to the abnormal cytogenetic changes as well as clinical features in childhood ALL.
Subject(s)
Adolescent , Child , Child, Preschool , Female , Humans , Infant , Male , Cytogenetics , Immunophenotyping , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Genetics , Allergy and Immunology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To look for the evidences of motilin receptor expression on interstitial cells of Cajal (ICC) of the rabbit.</p><p><b>METHOD</b>Smooth muscle segments with ICC were isolated from the small intestine of 10-day old rabbits. The tissue segments equilibrated in Ca(2+)-free Hanks' solution were dispersed with an enzyme solution containing collagenase type II and then Ficoll density centrifugation was used to dissociate ICC. The cells were suspended and cultured in the M199 medium. The c-kit antibody was applied to distinguish the cultured ICC. The motilin receptor was identified by immunocytochemical assay with GPR38 antibody, c-kit antibody and hoechst 33342 combined to label ICC. Cells cultured for a few days were sorted for ICC with c-kit stained green fluorescent through flow cytometry. The total RNA and proteins extracted from the sorted ICC were respectively used to verify motilin receptor on the ICC by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting.</p><p><b>RESULT</b>We had successfully dissociated and cultured ICC of rabbit small intestine in vitro. Fluorescent staining with c-kit antibody confirmed that the culture ICC was successful. Triple-labeled immunofluorescent staining had detected the motilin receptor on membrane of ICC. Flow cytometry analysis showed that the ratio of c-kit positive cell in the cultured cells was 64.3%. The number of sorted ICC was 6.7 × 10(5) and 5.6 × 10(6). The results of RT-PCR and Western blot confirmed that the ICC had motilin receptor expression.</p><p><b>CONCLUSION</b>Our study demonstrated presence of motilin receptor on ICC of the rabbit. The present results may suggest that ICC play an important role in gastrointestinal movement induced by motilin.</p>
Subject(s)
Animals , Rabbits , Cells, Cultured , Interstitial Cells of Cajal , Metabolism , Intestine, Small , Cell Biology , Receptors, Gastrointestinal Hormone , Metabolism , Receptors, Neuropeptide , MetabolismABSTRACT
<p><b>OBJECTIVE</b>The aim of this study was to investigate whether the induction of caspase-8 by γ-interferon (IFNγ) renders neuroblastoma (NB) cells sensitive to tumor necrosis factor related apoptosis inducing ligand(TRAIL).</p><p><b>METHODS</b>Caspase-8 mRNA expression was determined by RT-PCR. The effects of IFNγ, TRAIL, IFNγ +TRAIL and caspase-8 inhibitor+ TRAIL on the growth and apoptosis of NB cells were detected with the methods of reduction rate of Alamar Blue assay and flow cytometry. The relative caspase-8 activity was measured with colorimetric assay.</p><p><b>RESULTS</b>Caspase-8 expression was detectable in CHP212 cells which were sensitive to TRAIL, with an increased expression after treatment with IFNγ. Caspase-8 was undetectable in SH-SY5Y(SY5Y) cells which were resistant to TRAIL, but an increased expression of caspase-8 mRNA was found after treatment with IFNγ. Moreover, TRAIL combined with IFNγ induced apoptosis in SY5Y cells. The relative caspase-8 activity of CHP212 cells increased with the prolonged TRAIL action time. The relative caspase-8 activity of SY5Y cells in the IFNγ+TRAIL group was significantly higher than those of the control, IFNγ, TRAIL and inhibitor groups.</p><p><b>CONCLUSIONS</b>NB cells expressing caspase-8 are sensitive to TRAIL. TRAIL induces apoptosis in NB cells with an increase of relative caspase-8 activity.</p>
Subject(s)
Humans , Apoptosis , Caspase 8 , Physiology , Cell Line, Tumor , Flow Cytometry , Interferon-gamma , Pharmacology , Neuroblastoma , Drug Therapy , Pathology , Reverse Transcriptase Polymerase Chain Reaction , TNF-Related Apoptosis-Inducing Ligand , PharmacologyABSTRACT
The objective of this study was to investigate the immunophenotypic subtype profiles of 192 patients with acute myeloid leukemia (AML) and its association to cytogenetics and clinical features. Immunophenotyping of 192 patients was performed by flow cytometry using a panel of monoclonal antibodies. The karyotypes in 125 out of 192 cases were analyzed by G-banding technology. The results showed that CD33, CD13, myeloperoxidase (MPO) and CD117 were the most commonly expressed antigens in AML. CD117 expressed in 84.6% of AML-M3 cases. A combination of intensive autofluorescence, both CD34- and HLA-DR-, and high expression of CD13, CD33 and MPO had significant value for AML-M3 diagnosis. CD14 expressed only in AML-M4 and AML-M5, and both intensive positivity of CD64 and CD15 with high expression of HLA-DR may suggest great possibility for diagnosis of AML-M5. Lymphoid marker expression was documented in 47.9% of the 192 AML cases. CD56 (26.0%) and CD7 (20.8%) were the most commonly expressed lymphoid markers in AML patients, followed by CD19 (9.9%) and CD2 (7.3%). Abnormal karyotypes were detected in 76 out of 125 cases (60.8%). Correlation test showed that t(8;21) was found only in 17 cases of AML-M2 and strongly associated with the individual or combinational expressions of CD15/CD19/CD56. And 28 cases of t(15;17) were found in AML-M3; 2 cases of inv(16) were found in AML-M4EO. Higher CD34 positivity was found in LymAg+ group (77.2%) than that in LymAg- group (48.0%). It is concluded that immunophenotype analysis is useful for AML diagnosis and classification, and the immunophenotype has close relevance to the abnormal cytogenetic changes and clinical features in AML. The results suggested that a new prognostic scoring system that integrated the morphology, cytogenetic abnormalities and immunophenotype parameters would benefit the diagnosis, classification, and estimation of prognosis in AML patients.
Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Young Adult , Cytogenetics , Immunophenotyping , Leukemia, Monocytic, Acute , Genetics , Allergy and ImmunologyABSTRACT
This study was purposed to investigate the acute myeloid leukemia with complex karyotype t(2;21;8)(p12;q22;q22) (AML-M(2)) by using morphologic, immunologic, cytogenetic and molecular biologic classification technique (MICM) and to analyze the MICM characteristics of AML-M(2) and their diagnostic significance. The FAB typing of bone marrow cells (BMCs) was performed by Wright-Giemsa staining and histochemical staining of BM smears; the immunophenotype of leukemic cells was detected by flow cytometry; the karyotypes of chromosome samples prepared by short-term (48 hours) conventional culture of fresh BMCs were analyzed by RHG banding technique; the FISH signaling in mitotic metaphase was determined by dual color and dual fusion AML/ETO probe and chromosome painting probe, and was compared with results of conventional cytogenetic assay; the AML/ETO fusion transcripts were detected by nested RT-PCR. The results indicated that the bone marrow smears of case 1 showed extremely hyperplasia with myeloblasts in which a ratio of eosinophilic granulocytes and monocytes increased. Case 2 accorded with AML-M(2b) in which abnormal increase of myelocytes mainly appeared. The complex karyotype t(2;21;8)(p12;q22;q22) was detected by cytogenetic analysis combined with FISH in both two cases and AML1/ETO fusion transcripts were found by RT-PCR as well. The immunophenotype assay showed high co-expression of CD34 and HLA-DR accompanied with CD19 and CD56 expressions. It is concluded that application of MICM has an important significance for correct diagnostic typing of AML-M2 with complex karyotype variant of t(8; 21)(p12;q22;q22).
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Chromosomes, Human, Pair 2 , Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 8 , Immunophenotyping , Karyotyping , Leukemia, Myeloid, Acute , Diagnosis , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>Brain-derived neurotrophic factor (BDNF) and its specific tryrosin kinase receptor-B (TrkB) are highly correlated to the chemoresistance of neuroblastoma (NB) cells and poor prognosis. This study observed the changes of the sensibility of NB cells to chemotherapy drug cisplatin (CDDP) before and after blockage of TrkB-BDNF signal pathway by specific tyrosin kinase inhibitor K252a.</p><p><b>METHODS</b>Human NB cell line SH-SY5Y (SY5Y) was routinely cultured. Expression of TrkB was induced with nM all trans-retinoid acid (ATRA). Then BDNF, CDDP or K252a were added to the cultured SY5Y cells. Cell livability was assessed by methyl thiazolyl tetrazolium (MTT) assay. TrkB autophosphorylation was determined by Western blot analysis. Cell apoptosis rate was detected by flow cytometry (FCM). The conformation of apoptosis cells was observed by transmission electron microscopy (TEM).</p><p><b>RESULTS</b>The livability and apoptosis rate in SY5Y cells treated with ATRA, BDNF and CDDP were not different from the blank control group. However, after K252a together with ATRA, BDNF and CDDP treatment, the sensibility of SY5Y cells to chemotherapy drug CDDP increased, the livability decreased and the apoptosis rate increased in SY5Y cells when compared with the blank control group (P <0.01). K252a treatment resulted in blockage of TrkB autophosphorylation.</p><p><b>CONCLUSIONS</b>The blockage of TrkB-BDNF signal pathway by K252a use can increase sensibility of NB cells to chemotherapy and thus decrease the livability of NB cells.</p>
Subject(s)
Humans , Apoptosis , Brain-Derived Neurotrophic Factor , Carbazoles , Pharmacology , Cell Line, Tumor , Cisplatin , Pharmacology , Indole Alkaloids , Pharmacology , Microscopy, Electron, Scanning , Neuroblastoma , Drug Therapy , Pathology , Receptor, trkB , Signal Transduction , Tretinoin , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>To study the effect of gamma-interferon (IFNgamma), tumor necrosis factor related apoptosis inducing ligand (TRAIL), and cisplatin or etoposide induced apoptosis in human neuroblastoma cell line SH-SY5Y and its possible molecular mechanisms.</p><p><b>METHODS</b>The expressions of Caspase 8 mRNA and protein were detected with RT-PCR and Western blot analysis. The effects of IFN-gamma, TRAIL, IFNgamma + TRAIL, IFN-gamma + Caspase 8 inhibitor + TRAIL, IFNgamma + cisplatin + TRAIL, and IFNgamma + etoposide + TRAIL on the growth and apoptosis of SH-SY5Y cells were detected with the methods of MTT and flow cytometry. The relative Caspase 8 activity was measured with colorimetric assay.</p><p><b>RESULTS</b>Caspase 8 was undetectable in SH-SY5Y cells but an increased expression of Caspase 8 mRNA and protein was found after treatment with IFNgamma. SH-SY5Y cells themselves were not sensitive to TRAIL, but those expressing Caspase 8 after treatment with IFNgamma were. The killing effect of TRAIL on SH-SY5Y cells expressing Caspase 8 was depressed by Caspase 8 inhibitor. Cisplatin and etoposide could enhance the sensitivity of TRAIL on SH-SY5Y cells. The relative Caspase 8 activity of SH-SY5Y cells in IFN-gamma + TRAIL group was significantly higher than those of control group, IFN-gamma group, TRAIL group, and inhibitor group (P < 0.01). There was no significant difference among IFN-gamma + TRAIL group, IFNgamma + cisplatin + TRAIL group, and IFNgamma + etoposide + TRAIL group.</p><p><b>CONCLUSIONS</b>IFNgamma could sensitize SH-SY5Y cells to TRAIL-induced apoptosis and this may be realized by the up-regulation of Caspase 8. Cisplatin and etoposide could enhance the killing effect of TRAIL on SH-SY5Y cells.</p>
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Caspase 8 , Genetics , Cell Line, Tumor , Cell Survival , Cisplatin , Pharmacology , Etoposide , Pharmacology , Interferon-gamma , Pharmacology , Neuroblastoma , Genetics , Pathology , RNA, Messenger , Genetics , Reverse Transcriptase Polymerase Chain Reaction , TNF-Related Apoptosis-Inducing Ligand , PharmacologyABSTRACT
<p><b>OBJECTIVE</b>Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cell death in a variety of tumors but not in normal cells. TRAILdouble ended arrow-resistance of most neuroblastoma (NB) cell lines is related to the loss of caspase-8 expression and the expression and distribution of membrane TRAIL-receptors. This study investigated the role of caspase-8 and DR5 in TRAIL-induced apoptosis of NB cell line SKNDZ.</p><p><b>METHODS</b>The expression of caspase-8 mRNA was detected by RT-PCR. The expression of DR5 protein was detected by Western Blot analysis. The effects of TRAIL, IFNgamma +TRAIL, chemotherapeutic agent (adriamycin or etoposide) + TRAIL, and chemotherapeutic agent +TRAIL+ IFNgamma on the growth and apoptosis of SKNDZ cells were detected by MTT assay and flow cytometry.</p><p><b>RESULTS</b>caspase-8 was not expressed in SKNDZ cells but IFNgamma treatment resulted in an increase of caspase-8 expression. Expression of DR5 protein was not detected in SKNDZ cells but an increased DR5 protein expression was found after treatment with adriamycin or etoposide. The SKNDZ cells expressing caspase-8 were not sensitive to TRAIL but those SKNDZ cells expressing both caspase-8 and DR5 were sensitive. The early apoptosis rates of the adriamycin /etoposide + IFNgamma+TRAIL groups [(17.9 +/- 3.6)%, (14.8 +/- 3.3)%] were higher than that of the IFNgamma+TRAIL group [(3.9 +/- 1.2)% ](F=26.233, P < 0.01).</p><p><b>CONCLUSIONS</b>SKNDZ cells expressing both caspase-8 and DR5 restored the TRAIL sensitivity. Caspase-8 and DR5 play a key role in TRAIL-induced apoptosis of NB cells.</p>